Project description:The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5’ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5’ and 3’ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression. Whole-genome bisulfite sequencing of 24-hour adult female Nasonia vitripennis whole body samples using Iilumina GAIIx and HiSeq2000.

Project description:The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5’ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5’ and 3’ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.

Project description:Background The generalist dipteran pupal parasitoid Nasonia vitripennis injects 79 venom peptides into the host before egg laying. This venom induces several important changes in the host, including developmental arrest, immunosuppression, and alterations to normal metabolism. It is hoped that diverse and potent bioactivities of N. vitripennis venom provide an opportunity for the design of novel acting drugs. However, currently very little is known about the individual functions of N. vitripennis venom peptides and less than half can be bioinformatically annotated. The paucity of annotation information complicates the design of studies that seek to better understand the potential mechanisms underlying the envenomation response. Although the RNA interference system of N. vitripennis provides an opportunity to functionally characterise venom encoding genes, with 79 candidates this represents a daunting task. For this reason we were interested in determining the expression levels of venom encoding genes in the venom gland, such that this information could be used to rank candidate venoms. To do this we carried out deep sequencing of the transcriptome of the venom gland and neighbouring ovary tissue and used RNA-seq to measure expression from the 79 venom encoding genes. The generation of a specific venom gland transcriptome dataset also provides further opportunities to investigate novel features of this highly specialised organ. Results High throughput sequencing and RNA-seq revealed that the highest expressed venom encoding gene in the venom gland was a serine protease called Nasvi2EG007167, which has previously been implicated in the apoptotic activity of N. vitripennis venom. As expected the RNA-seq confirmed that the N. vitripennis venom encoding genes are almost exclusively expressed in the venom gland relative to the neighbouring ovary tissue. Novel peptides appear to perform key roles in N. vitripennis venom function as only four of the highest 15 expressed venom encoding genes are bioinformatically annotationed. The high throughput sequencing data also provided evidence for the existence of an additional 471 novel genes in the Nasonia genome that are expressed in the venom gland and ovary. Finally, metagenomic analysis of venom gland transcripts identified viral transcripts that may play an important part in the N. vitripennis venom function. Conclusions The expression level information provided here for the 79 venom encoding genes provides an unbiased dataset that can be used by the N. vitripennis community to identify high value candidates for further functional characterisation. These candidates represent bioactive peptides that have value in drug development pipelines.

Project description:The extraordinary range in the degree of sexual dimorphism (SD) among animal species is widely perceived to be caused in part by differences in patterns of sexual selection, but sex-specific adaptations and sex chromosome differences also play a role. Studies in insects have discovered a substantial number of sex-biased genes, but little is known about the epigenetic basis of SD. The degree and genome-wide distribution of sex-biased expression become interesting questions in hymenoptera species with haplodiploid sex-determination. To study the genetic and epigenetic architecture of SD and understand the conservation and evolution of sex-biased expression in a haplodiploid system that lacks sex chromosomes, we performed RNA-seq and whole-genome bisulfite sequencing in female and male adult samples of two parasitoid wasp species, Nasonia vitripennis and Nasonia giraulti. More than 75% of the expressed genes displayed significantly sex-biased expression. Both the number and the degree of sex-biased genes are higher than insects like Drosophila melanogaster, which have sex-chromosome mediated sex determination. Females from the two Nasonia species have far more similar expression profiles than does the contrast between the two sexes within either species. Interestingly, the extremely male- and female-biased genes are enriched for totally different functional categories: male-biased genes are highly enriched for key enzymes in sex-pheromone synthesis; female-biased genes are enriched for nuclear-located genes that are responsible for epigenetic regulation of gene expression. Unlike gene expression profiles, DNA methylomes are more similar within species, and no stable differentially methylated genes have been found between the two sexes, suggesting that DNA methylation is not directly responsible for the molecular basis of SD. However, methylation status does influence sex-biased expression: 80% of female-biased genes are methylated, which is more than two-fold higher than the genome average (30%); almost all male-biased and sex-specific genes are non-methylated, which is consistent with the fact that methylated genes have house-keeping functions and a broader expression breadth. Evolutionarily, male-biased genes have greater sequence divergence between the two species, and they are more likely to have a functional paralog in the Nasonia genome. Sex-specific genes have significantly higher non-synonymous substitution rates and dN/dS ratios. In addition, local clusters of sex-biased genes in the genome may have epigenetic properties similar to the sex chromosome. In summary, Nasonia accomplish a striking degree of sex-differential expression through a difference in ploidy along with associated differences in methylations status. Whole-genome bisulfite sequencing of 24-hour adult whole body samples of Nasonia vitripennis and Nasonia giraulti using Iilumina sequencing.

Project description:The extraordinary range in the degree of sexual dimorphism (SD) among animal species is widely perceived to be caused in part by differences in patterns of sexual selection, but sex-specific adaptations and sex chromosome differences also play a role. Studies in insects have discovered a substantial number of sex-biased genes, but little is known about the epigenetic basis of SD. The degree and genome-wide distribution of sex-biased expression become interesting questions in hymenoptera species with haplodiploid sex-determination. To study the genetic and epigenetic architecture of SD and understand the conservation and evolution of sex-biased expression in a haplodiploid system that lacks sex chromosomes, we performed RNA-seq and whole-genome bisulfite sequencing in female and male adult samples of two parasitoid wasp species, Nasonia vitripennis and Nasonia giraulti. More than 75% of the expressed genes displayed significantly sex-biased expression. Both the number and the degree of sex-biased genes are higher than insects like Drosophila melanogaster, which have sex-chromosome mediated sex determination. Females from the two Nasonia species have far more similar expression profiles than does the contrast between the two sexes within either species. Interestingly, the extremely male- and female-biased genes are enriched for totally different functional categories: male-biased genes are highly enriched for key enzymes in sex-pheromone synthesis; female-biased genes are enriched for nuclear-located genes that are responsible for epigenetic regulation of gene expression. Unlike gene expression profiles, DNA methylomes are more similar within species, and no stable differentially methylated genes have been found between the two sexes, suggesting that DNA methylation is not directly responsible for the molecular basis of SD. However, methylation status does influence sex-biased expression: 80% of female-biased genes are methylated, which is more than two-fold higher than the genome average (30%); almost all male-biased and sex-specific genes are non-methylated, which is consistent with the fact that methylated genes have house-keeping functions and a broader expression breadth. Evolutionarily, male-biased genes have greater sequence divergence between the two species, and they are more likely to have a functional paralog in the Nasonia genome. Sex-specific genes have significantly higher non-synonymous substitution rates and dN/dS ratios. In addition, local clusters of sex-biased genes in the genome may have epigenetic properties similar to the sex chromosome. In summary, Nasonia accomplish a striking degree of sex-differential expression through a difference in ploidy along with associated differences in methylations status. Profiling of expression levels in Nasonia vitripennis and Nasonia giraulti adult male and female samples using Illumina RNA-seq

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Profiling of expression level in adult female Nasonia vitripennis

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